Cancer is a leading cause of death in the world and elsewhere. Depending on the type of cancer, it is typically treated with surgery, chemotherapy, and/or radiation. These treatments often fail: surgery may not remove all the cancer and metastases may occur; some cancers are resistant to chemotherapy and radiation therapy; and chemotherapy-resistant tumors frequently develop. New therapies are necessary, to be used alone or in combination with classical techniques.
One potential therapy under active investigation is treating tumors with recombinant viral vectors expressing anti-cancer therapeutic proteins. Adenoviral vectors (Ad) have proven to be of enormous utility for a variety of gene therapy applications. Adenovirus-based vectors contain several characteristics that make them conceptually appealing for use in treating cancer, as well as for therapy of genetic disorders. Adenoviruses can easily be grown in culture to high titer stocks that are stable. They have a broad host range, replicating in most human cancer cell types. Their genome can be manipulated by site-directed mutation and insertion of foreign genes regulated by foreign promoters.
Conditionally-replicative adenoviruses (CRAds) have been a novel class of anticancer agents, which are designed to selectively replicate in tumor cells and to lyse them (1-3). U.S. Pat. No. 7,109,029 discloses a viral vector, which has at least one interfering genetic element, comprising at least one transcription unit, wherein at least one insulating sequence is located 5′ to the transcription initiation site of said transcription unit and 3′ to said interfering genetic element. U.S. Pat. No. 7,026,164 provides adenovirus packaging cell lines for growth of an E1A/E1B deficient adenovirus that is substantially free of replication competent adenovirus (RCA). CRAd-based cancer treatments are already being evaluated in clinical trials (4-7). The safety and efficacy of CRAds depend on the specific viral replication in tumor versus in normal cells. Deleting viral genes encoding proteins required for the viral life cycle in normal cells but not in tumor cells is a strategy to induce a tumor-specific viral replicative lysis (3). This strategy has been exploited in E1B-55 kD-deleted therapeutic CRAds, which were designed to exert specific cytopathic effect on p53-nonfunctional tumor cells (8, 9). The p53 gene is one of the most studied and well-known genes. p53 plays a key role in cellular stress response mechanisms by converting a variety of different stimuli, for example, DNA damage, deregulation of transcription or replication, and oncogene transformation, into cell growth arrest or apoptosis. p53 is inactivated in a majority of human cancers. When p53 is inactivated, abnormal tumor cells are not eliminated from the cell population, and are able to proliferate. An elegant example of E1B-55 kD-deleted therapeutic CRAds is ONYX-015 (dl1520), which has shown definitive antitumour activities in p53 nonfunctional tumors (10, 11). The E1B-55 kD-deleted Ads failed to replicate efficiently in cells with p53wild. Induction of p53 expression by E1B-55 kD-deleted Ads in normal cells is deemed to be contributory for the resistance of these cells to lytic replication of the E1B-55 kD-deleted Ads. Conversely, the lack of p53 accounts for the permissiveness of tumor cells to the lytic replication of E1B-55 kD-deleted Ads.
The human ras gene family consists of three members: the H-ras, K-ras and the N-ras gene. These genes code for related proteins of 21 kD, which are located at the inner face of the cell membrane and are thought to be involved in transducing signals from cell surface receptors to their intracellular targets. A significant portion of tumor cell lines and fresh tumor tissue has been found to possess an activated ras gene. Such genes are characterized by their ability to induce oncogenic transformation of cells. In most cases so far analyzed the activation is due to a point mutation in the 12nd or 13rd codon of a ras gene resulting in a single amino acid substitution in the gene product. Activating mutation of RAS has been implicated in tumorigenesis of many malignancies including lung cancer and colorectal cancer at rates of around 21% and 34%, respectively (12, 13). There were also many other malignancies harboring RAS activating mutations and exhibiting elevated RAF activities (14). With an aim to extend the cytopathic spectrum of CRAds to tumors with activating RAS, a strategy taking advantage of conditional sequestering of p53 in tumor cells but leave unaffected bystander normal cells bearing p53wild may be used. Paradoxically, it was found that an E1B-55 kD-deleted Ad could replicate in p53wild tumor cells (15). In these cases, Hdm2, the negative regulator of p53 might be implicated for it is transcriptionally upregulated by p53 and forms with p53 a feedback loop to inactivate p53 (16). Noticeably, the transcription of hdm2 gene could also be turned on by the RAS-upregulated RAF/MEK/MAPK pathway, in a p53-independent manner (17). A Ras/Raf signaling cascade-responsive element composed of ETSA and AP-1/ETSB elements was identified in the mouse double minute 2 (mdm2) P2 promoter, just upstream of the p53 responsive promoter elements (17, 18). The hdm2 P2 promoter is composed of AP-1/ETSa, which are the conserved homologues of AP-1/ETSB in mdm2 P2 promoter; however, does not include the counterpart of ETSA element in mdm2 P2 promoter. Nonetheless, Ras/Raf signaling cascade-responsiveness of the hdm2 P2 promoter was indeed found in human cancer cells (17).
Thus, there is a continuing need for vectors that replicate and spread efficiently in tumors but that can be modified such that they replicate poorly or not at all in normal tissue.